Project Summary Nitrogen fixation (N2 reduction) represents the most significant input of nitrogen into the biogeochemical nitrogen cycle. The biological catalyst for nitrogen fixation is called nitrogenase, an enzyme that is only produced by certain microorganisms called diazotrophs. Nitrogenase contains two component proteins, one of these is a nucleotide-dependent agent of electron delivery (called the Fe protein) and the other (called MoFe protein) contains the site for substrate binding and reduction. The work proposed herein seeks to address some of the key unanswered questions about the nitrogenase catalytic mechanism. During the last funding cycle, methods were developed that allow the normal substrate N2, as well as proposed reduction intermediates, diazene (HN=NH and its homolog methydiazene HN=N-CH3), and hydrazine (H2N-NH2) to be trapped in high concentrations within the MoFe protein. Preliminary studies have revealed these trapped species are bound to a complex organo-metallocluster, called FeMo-cofactor [7Fe-9S-Mo-X-homocitrate], which provides the nitrogenase active site. Each of the trapped states is paramagnetic thereby permitting the application of electron paramagnetic resonance (EPR) and enhanced nuclear double resonance (ENDOR) spectroscopies as powerful probes to deduce molecular details of the bound species. In the first aim, we seek to characterize each of these trapped states as an approach to explore intermediate states that occur during the catalytic reduction of N2. Studies proposed include optimization of the N2-trapped state, and a temperature step-annealing strategy to connect the intermediates to each other and to the mechanism. Collectively, these studies are expected to allow development of an experimentally driven model that describes the nature of the catalytic intermediates. A strategy is presented in the second aim that will probe a putative substrate channel within the MoFe protein component. This channel is proposed to provide a pathway for substrate movement from the protein surface to FeMo-cofactor. For these studies, amino acids that line the proposed channel within the MoFe protein will be substituted individually and in combinations and the impact of these substitutions on kinetic parameters for reduction of a range of substrates of varying size will be determined. X-ray crystal structures of the most interesting MoFe proteins will also be pursued. It is expected that these studies will provide the first experimental evidence that define a specific substrate channel within nitrogenase. The P-cluster is a second complex metallocluster ([8Fe-7S]) contained within the MoFe protein that is proposed to function in the intermolecular delivery of electrons to FeMo-cofactor. Studies presented in the third aim seek to define the role of the P-cluster in electron transfer during the catalytic cycle. We have developed a freeze-quench EPR spectroscopic approach that allows simultaneous observation of changes in the oxidation states of the P-cluster and FeMo-cofactor during the catalytic cycle. Preliminary results reveal that electron transfer from the P-cluster to FeMo-cofactor can be controlled and monitored by using a temperature step-annealing technique that avoids the confounding continuous addition of electrons from the Fe protein. These studies offer the first opportunity to monitor internal electron transfer within nitrogenase and are therefore expected to provide significant new insights into the role of the P-cluster. The fourth aim seeks to reveal the identity of X, the unknown atom that is located at the center of FeMo-cofactor. Preliminary ENDOR spectroscopic results reveal a strongly coupled N-atom in the wild-type MoFe protein trapped during turnover with N2 bound. The strongly coupled N is not coming from the bound N2. An experimental approach is presented to identify the origin of this strongly coupled N atom (possibly X) by using a MoFe protein assembled from an apo- MoFe protein labeled with either 14N or 15N and isolated FeMo-cofactor labeled with either 14N or 15N. Resolving the identity of X is important as it will provide mechanistic insight with respect to the electronic structure of the nitrogenase active site, it will impact the application of computational methods that seek to model the catalytic process, and it will provide information that is necessary to understand how FeMo-cofactor is formed biologically and how it might be produced synthetically. PUBLIC HEALTH RELEVANCE: Narrative The majority of N2 reduction that occurs today, ultimately supporting the nitrogen demand of an estimated 60% of the human population, is by biological nitrogen fixation within microorganisms, called diazotrophs. The catalyst for this N2 reduction is the enzyme nitrogenase. In this work, we seek to deduce key aspects of the mechanism of nitrogenase.